Axonopathy is an early and prominent pathological feature of many central nervous system (CNS) disorders, including brain and spinal cord trauma, optic neuropathy, Multiple Sclerosis, early stage Alzheimer?s disease, and subcortical ischemia. Poor clinical outcomes in all of these neurological conditions are due, in large part, to the limited regenerative capacity of adult CNS neurons, including retinal ganglion cells (RGC, the neurons that give rise to the optic nerve). There is a dire need to develop novel therapeutic interventions that overcome barriers to repair in the adult CNS and promote axonal regrowth. The studies proposed here are based on our discovery of a novel subset of pro-regenerative neutrophils, characterized by the cell surface phenotype Ly6GlowCD14+, that accumulate in the posterior chamber of the eye or the peritoneal cavity following local administration of the yeast cell wall extract, zymosan. These neutrophils bear a ring-form nucleus and express high levels of pattern recognition receptor, dectin-1, as well as transcripts for arginase-1 and CD206. In preliminary studies we demonstrated that adoptive transfer of zymosan-elicited Ly6GlowCD14+ neutrophils directly into the vitreous of mice with optic nerve crush (ONC) injury is sufficient to rescue RGC from cell death and to stimulate the regrowth of severed RGC axons. Furthermore, conditioned media harvested from cultures of Ly6GlowCD14+neutrophils induce neurite outgrowth of dissociated RGC and dorsal root ganglion neurons in vitro. The overall goal of the current proposal is to elucidate the pathways that underlie the differentiation, survival and mechanism of action of these unconventional reparative neutrophils, and to leverage the knowledge gained for the development of immunomodulatory therapies that mitigate, or even reverse, damage to CNS neurons and axons. In Aim 1 we will test our hypothesis that transforming growth factor (TGF)-? drives the differentiation of Ly6GlowCD14+ neutrophils in vivo following the administration of zymosan. In Aim 2 we will determine the role of hypoxia induced factor (HIF)-1? in the stabilization, survival and biological functions of Ly6GlowCD14+ neutrophils. In Aim 3 we will optimize protocols for the generation of pro-regenerative neutrophils from bone marrow precursors ex vivo. Selected neutrophil lines will be infused into mice with ONC injury to assess their efficacy as an autologous cellular therapy. In addition, we will use proteomic and genetic approaches to characterize the soluble factors present in Ly6Glow neutrophil-conditioned media that are responsible for enhanced neurite outgrowth. A future direction will be to administer candidate neuroregenerative factors to mice with axonopathy as disease modifying agents. We are hopeful that the data generated by our study will ultimately lead to the development of innovative cell based therapies and/ or immunomodulatory drugs with neuroprotective/ regenerative properties that restore lost neurological functions in patients with CNS trauma or other conditions characterized by axonopathy.